Manual Microseparation And TCR Gene Cloning Of Single T Lymphocytes

Objective:To establish the isolation and amplification technique of single T cell for obtaining the TCR gene that recognizes the specific epitope peptide,providing a new method for the acquisition of the TCR that identifies tumor and laying the foundation for adoptive immunotherapy of specific TCR gene-modified T cells.Methods:1.Study on single-lymphocyte manual microisolation and single-cell RT-PCRSingle T lymphocytes were isolated by this self-made tool assembled with the capillary glass tube,rubber tube and filter and then with different kits for reverse transcription and GAPDH gene amplification.The reverse transcription kit with high amplification efficiency was determined and the amplification efficiency of BCL2,STAT1 and CD8a genes was further verified and further optimize the corresponding amplification conditions,which laid the foundation for the amplification of TCR genes of individual T cells.2.The establishment of the method of single-cell amplification of TCR geneAfter single enriched CD8~+T cell was isolated by a self-made single-cell manual isolation tool,the TCRA genes of single T cell were amplified using the 5'RACE method and the multiplex PCR method,respectively.Different conditions for amplification,different specific primers,and different PCR amplification enzymes were used to explore the conditions for the amplification of the TCRA gene using the 5'RACE method;TCRA gene amplification was performed by conventional multiplex PCR and step-by-step multiplex PCR for establishing a stably TCRA amplification method on single-cell level.3.The construction of TCR(?and?)recombinant expression vectorsThe TCR??core sequences(CDR3)obtained by single cell amplification in the second part were spliced to the full-length sequences by fusion PCR with the corresponding V and C region fragments,respectively.Then TCR?and TCR?gene fragments were amplified by designing appropriate homologous recombination primers and restriction enzyme primers based on the inserted target genes and vector sequence,and then were ligated with the pIRES2-Oligo vector to construct TCR recombinant expression vector.Result:1.First,the successful assembled of single-cell separation tool and a method for artificial microscopic separation of single T lymphocytes was completed;Then a stable single cell transcription and amplification method was explored by the amplification of the GAPDH gene with a single cell and it has been further validated by amplifying BCL2,STAT1,and CD8a genes from a single cell,laying the foundation for the amplification of TCR genes in individual T cells.2.The 5'RACE method failed to amplify the TCRA gene of a single T cell.Then after the optimization of multiplex PCR amplification of single T cell TCRA gene,two T cell TCRA genes were successfully amplified:TCR V?16 and TCR V?2/3 from T cell of No.1 and TCR V?26 and TCR V?1/5 from T cell of No.4.3.The full-length sequences of TCR?5,TCR?1/5 and TCR?26 were successfully amplified by fusion PCR.pIRES2-TCR?5-TCR?1/5 and pIRES2-TCR?5-TCR?26 Recombinant expression vectors were successfully constructed by homologous recombination and enzyme digestion.Conclusions:In this experiment,a single-cell manual microseparation and gene amplification method of T cells were successfully established.Based on this method,a single-cell TCR gene amplification method was explored and a method of stepwise multiplex PCR amplification of TCR genes on the single-cell level was successfully established.The establishment of this technology provides a new method and idea for the rapid acquisition of specific TCR genes required for TCR-T cell immunotherapy,laying the foundation for promoting the application of TCR-T adoptive cell immunotherapy.